Method of treating lung disease with uridine triphosphates

ABSTRACT

A method of hydrating mucous secretions in the lungs of a subject in need of such treatment is disclosed. The method comprises administering to the lungs of the subject a uridine triphosphate such as uridine 5&#39;-triphosphate (UTP) or uridine 5&#39;-O-(3-thiotriphosphate) (UTPγS) in an amount effective to hydrate lung mucous secretions. The method is useful for treating patients aflicted with cystic fibrosis. Pharmaceutical formulations and methods of making the same are also disclosed.

This invention was made with Government support under Grants HL34322 andHL42384 from the National Institutes of Health. The Government may havecertain rights to this invention.

FIELD OF THE INVENTION

This invention relates to a method of removing retained mucus secretionsfrom the lungs of a patient by administering certain uridinetriphosphates to the lungs of the patient.

BACKGROUND OF THE INVENTION

Extracellular adenosine triphosphate has been shown to regulate avariety of biological processes including non-vascular smooth musclecontraction (M. Maguire and D. Satchell, J. Pharmacol. Exp. Ther. 211,626-631 (1979); C. Brown and G. Burnstock, Eur. J. Pharmacol. 69, 81-86(1981)) and vascular tone (G. Burnstock and C. Kennedy, Circ. Res. 58,319-330 (1986); D. Haeussinger et al., Eur. J. Biochem. 167, 65-71(1987)), platelet aggregation (G. Born and M. Kratzer, J. Physiol.(Lond.) 354, 419-429 (1984)), neurotransmission (G. Burnstock, Nature,229, 282-283 (1971); G. Burnstock and P. Sneddon, Clin. Sci. 68 (Suppl.10), 89s-92s (1985)), and cellular ion transport (G. Burgess et al.,Nature 279, 544-546 (1979); D. Galacher, Nature 296, 83-86 (1982)) andsecretory activities (J. Chapal and M-M. Loubatieres-Mariani, Br. J.Pharmacol. 73, 105-110 (1981); J. Pearson et al., Biochem. J. 214,273-276 (1983)). These effects are mediated by specific purinergicreceptors which respond to ATP or other nucleotides present in theextracellular millieu (J. Gordon, Biochem. J. 233, 309-319 (1986)).

Purinoceptors have been functionally identified in rat pulmonaryepithelia in studies of regulation of alveolar Type II surfactantphospholipid secretion (W. Rice and F. Singleton, Br. J. Pharmacol. 89,485-491 (1986)). To our knowledge these receptors have not been reportedin human airway epithelial cells. Because ion transport appears to beregulated by purinergic receptor stimulation in other epithelia (Burgesset al., supra (1979); Gallacher, supra (1982)), we investigated severalfeatures of the effect of extracellular nucleotides on the ion transportactivities of human airway epithelium.

Purinergic receptor regulation of ion transport might have potentialtherapeutic benefit in lung diseases characterized by abnormalities inepithelial ion transport, e.g., cystic fibrosis. In cystic fibrosis theairway epithelial dysfunction is expressed in part by defectiveregulation of Cl⁻ ion transport by secretagogues that regulate theapical cell membrane Cl⁻ channel by cAMP-dependent or protein kinase Cdependent mechanisms (R. Boucher et al., J. Clin. Invest. 78, 1245-1252(1986); R. Boucher et al., J. Clin. Invest. 84, 1424-1431 (1989); J.Riordan et al., Science 245, 1066-1073 (1989); J. Rommens et al.,Science 245, 1059-1065 (1989)). Induction of Cl⁻ secretion by CF airwayepithelia in vivo might help liquify the relatively dehydrated, thickairway surface liquid that characterizes this disease. We thereforetested whether nucleotides would bypass regulatory defects in CF airwayepithelia and induce Cl⁻ secretion at rates similar to those of normalairway cells. The present invention is based upon this investigation.

SUMMARY OF THE INVENTION

A method of hydrating mucous secretions in the lungs of a subject inneed of such treatment is disclosed. The method comprises administeringto the lungs of the subject a compound of Formula I below, or apharmaceutically acceptable salt thereof (hereinafter referred to as the"active compound"), in an amount effective to hydrate lung mucoussecretions: ##STR1## wherein:

X₁, X₂, and X₃ are each independently either O⁻ or S⁻. Preferably, X₂and X₃ are O⁻.

R₁ is O, imido, methylene, or dihalomethylene (e.g., dichloromethylene,difluoromethylene). Preferably, R₁ is oxygen.

R₂ is H or Br. Preferably, R₂ is H. Particularly preferred compounds ofFormula (I) above are uridine 5'-triphosphate (UTP) and uridine5'-O-(3-thiotriphosphate) (UTPγS).

The method of the present invention may further comprise the step ofconcurrently administering amiloride to the subject in an amounteffective to inhibit the reabsorption of water from lung mucoussecretions.

A second aspect of the present invention is a pharmaceutical formulationcontaining the active compounds disclosed herein, in an amount effectiveto hydrate lung mucous secretions, in a pharmaceutically acceptablecarrier. The pharmaceutical formulation may further contain amiloride inan amount effective to inhibit the reabsorption of water from lungmucous secretions.

A third aspect of the present invention is the use of the activecompounds disclosed herein for the manufacture of a medicament for thetherapeutic hydration of mucous secretions in the lungs of a patient inneed of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the log concentration-effect curves (percent change inI_(sc) from basal levels) of nucleotides applied to the basolateralsurface of normal human nasal epithelium. s.e. of each data point is≦15% of the normalized maximum response.

FIG. 2 shows the log concentration-effect curves (percent change inI_(sc) from basal levels) of nucleotides applied to the apical surfaceof human nasal epithelium pretreated with amiloride (10⁻⁴ M). s.e. ofeach data point is ≦13% of the normalized maximum resonse.

FIG. 3A shows, in log concentration-effect on [CA²⁺ ]_(i), a comparisonof agonists which bind P_(2x), P_(2y) or UTP sensitive receptors, meanbasal [CA²⁺ ], in each set of experiments were (in nM): UTP (◯), 81±8,range 48-113, n=5 (n=3-6); ATPτS ( ), 116±11, range 85-151, n=4 (n=3-6);ATP (□), 61±3, range 51-70, n=8 (n=3-8 at each agonist concentration);2MeSATP ( ), 123±19 range 95-180, n=3 (n=3); ADPβS (Δ), 91±17, range74-107, n=3 (n=3); αβMeATP ( ), 82±16, range 50-99, n=3 (n=3); βγMeATP (), 68±4, range 61-74, n=3 (n=3).

FIG. 3B shows, in log-concentration effect on [Ca²⁺ ]_(i), a comparisonof other purine agonists with response stimulated by ATP (◯), mean basal[Ca²⁺ ], in each set of experiments were (nM): GTP ( ), 117±13, range92-153, n=3 (n=3; AMPPNP (□), 137±50, range 74-235, n=3 (n=3; ITP ( ),103±10, range 85-121, n=3 (n=3); ADP (Δ), 94±16, range 75-127, n=3(n=2-3); AMP ( ), 111±28, range 69-189, n=3 (n-3); ATPαS ( ), 89±2,range 87-91, n-1 (n=2).

FIG. 3C shows, in log-concentration effect on [Ca²⁺ ]_(i), a comparisonof UTP (◯) with other pyrimide agonists, mean basal [Ca²⁺ ], (in nM):5BrUTP ( ), 110±13, range 82-152, n=3 (n=2-3); UDP (□), 89±2, range84-92, n=3 (n=3); CTP (Δ), 83±10, range 69-102, n=3 (n=3); UMP ( ),78±14, range 64-91, n=2 (n=2).

FIG. 4A provides a bioelectric tracing of the effect on I_(sc) ofextracellular ATP (10⁻⁴ M) applied to the apical surface ofamiloride-pretreated (10⁻⁴ M) CF human nasal epithelium, showing Cl⁻secretion in response to ATP (post-amiloride I_(sc) =13 μA.cm⁻²).

FIG. 4B is a bioelectric tracing like FIG. 4A showing the I_(sc)response to ATP (10⁻⁴ M) of opposite polarity (post-amiloride I_(sc) =19μA.cm⁻²).

FIG. 4C is a bioelectric tracing like FIG. 4A showing the Cl⁻ secretoryresponse to UTP (10⁻⁶ M) (post-amiloride I_(sc) =5 μA.cm⁻²).

FIG. 5 shows the log concentration-effect curves for changes in I_(sc)from basal levels when ATP or UTP are applied to the apical surface ofamiloride-pretreated CF tissues. s.e. of each data point is ≦13% of thenormalized maximum response.

FIG. 6 shows the log concentration-effect relationships of the effect ofATP and UTP on [Ca²⁺ ]_(i) (change from basal levels) in single CF nasalepithelial cells. s.e. of each data point is ≦8% of the normalizedmaximum response.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention may be used to hydrate mucoussecretions in the lungs of a subject in need of such treatment for anyreason, including (but not limited to) retained secretions arising fromairway diseases such as cystic fibrosis, chronic bronchitis, asthma, andbronchiectasis. Hydration of the mucous secretions causes allows them tobe more easily transported from the lungs via mucociliary action, andhence facilitates the removal of retained mucous secretions.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

Compounds illustrative of the compounds of Formula (I) above include:(a) uridine 5'-triphosphate (UTP); (b) uridine 5'-O-(3-thiotriphosphate) (UTPγS); and (c) 5-bromo-uridine 5'-triphosphate(5-BrUTP). These compounds are known or may be made in accordance withknown procedures, or variations thereof which will be apparent to thoseskilled in the art. See generally N. Cusack and S. Hourani, Annals N.Y.Acad. Sci. 603, 172-181 (G. Dubyak and J. Fedan Eds. 1990) (titled"Biological Actions of Extracellular ATP"). For example, UTP may be madein the manner described in Kenner et al., J. Chem. Soc. 1954, 2288; orHall and Khorana, J. Chem. Soc. 76, 5056 (1954). See Merck Index,Monograph No. 9795 (11th Ed. 1989). UTPγS may be made in the mannerdescribed in G. Goody and F. Eckstein, J. Am. Chem. Soc. 93, 6252(1971).

For simplicity, Formula I herein illustrates uridine triphosphate activecompounds in the naturally occuring D configuration, but the presentinvention also encompasses compounds in the L configuration, andmixtures of compounds in the D and L configurations, unless specifiedotherwise. The naturally occuring D configuration is preferred.

The active compounds disclosed herein may be administered to the lungsof a patient by any suitable means, but are preferably administered byadministering an aerosol suspension of respirable particles comprised ofthe active compound, which the subject inhales. The respirable particlesmay be liquid or solid. The particles may optionally contain othertherapeutic ingredients such as amiloride, with amiloride included in anamount effective to inhibit the reabsorption of water from airway mucoussecretions, as described in U.S. Pat. No. 4,501,729 (applicantspecifically intends the disclosure of this and all other patentreferences cited herein be incorporated herein by reference). The term"amiloride" as used herein, includes the pharmaceutically acceptablesalts thereof, such as (but not limited to) amiloride hydrochloride. Thequantity of amiloride included may be an amount sufficient to achievedissolved concentrations of amiloride on the airway surfaces of thesubject of from about 10⁻⁷ to about 10⁻³ Moles/liter, and morepreferably from about 10⁻⁶ to about 10⁻⁴ Moles/liter.

The active compounds disclosed herein can be prepared in the form oftheir pharmaceutically acceptable salts. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (b) salts formed fromelemental anions such as chlorine, bromine, and iodine.

Particles comprised of active compound for practicing the presentinvention should include particles of respirable size: that is,particles of a size sufficiently small to pass through the mouth andlarynx upon inhalation and into the bronchi and alveoli of the lungs. Ingeneral, particles ranging from about 1 to 10 microns in size (moreparticularly, less than about 5 microns in size) are respirable.Particles of non-respirable size which are included in the aerosol tendto deposit in the throat and be swallowed, and the quantity ofnon-respirable particles in the aerosol is preferably minimized.

Liquid pharmaceutical compositions of active compound for producing anaerosol may be prepared by combining the active compound with a suitablevehicle, such as sterile pyrogen free water. Other therapeutic compoundssuch as amiloride may optionally be included.

Solid particulate compositions containing respirable dry particles ofmicronized active compound may be prepared by grinding dry activecompound with a mortar and pestle, and then passing the micronizedcomposition through a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprised of the activecompound may optionally contain a dispersant which serves to facilitatethe formation of an aerosol. A suitable dispersant is lactose, which maybe blended with the active compound in any suitable ratio (e.g., a 1 to1 ratio by weight). Again, other therapeutic compounds such as amiloridemay also be included.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on theairway surfaces of the subject of from about 10³¹ 7 to about 10⁻³Moles/liter, and more preferably from about 10⁻⁶ to about 3×10⁻⁴Moles/liter. Depending upon the solubility of the particular formulationof active compound administered, the daily dose may be divided among oneor several unit dose administrations.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer. See U.S. Pat. No. 4,501,729(applicant specifically intends that the disclosure of this and allother patent references cited herein be incorporated herein byreference).

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing a predetermined metered dose ofa medicament at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator comprises achamber having a rotor mounted therein, which rotor carries a gelatincapsule containing a metered dose of dry particle medicament. In use thecapsule is pierced, a patient inhales through the chamber, and the rotoris caused to spin at a speed sufficient to dispense the medicament tothereby form an aerosol of dry particles. A second type of illustrativeaerosol generator comprises a pressurized canister containing dryparticle medicament in a propellant. The propellant is dischargedthrough a metering valve configured to dispense a metered dose of thedry particle medicament into the atmosphere. The propellant evaporates,leaving an aerosol of dry particle medicament.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferably from about 30 to 150 liters perminute, and most preferably about 60 liters per minute. Aerosolscontaining greater amounts of medicament may be administered morerapidly.

The present invention is explained in greater detail in the Exampleswhich follow. These examples are intended as illustrative of theinvention, and are not to be taken as limiting thereof. ATP, UTP, ATPγS,CTP, GTP, ITP, adenylyl-imidodiphosphate (AMPPNP), β,γ-methylene ATP(β,γ-MeATP), ADPβS, ADP, AMP, UDP, 5-BrUTP, UMP, ATPαS and dipyridamolewere obtained from Boehringer Mannheim Biochemicals (Indianapolis,Ind.). α,β-methylene ATP (α,β-Me ATP) and amiloride were obtained fromSigma Chemicals (St. Louis, Mo.). 2-methylthio ATP (2MeSATP) waspurchased from Research Biochemicals Inc.(Natick, Mass.). Bioelectricproperties were measured with confluent monolayers bathed by Ham's F-12culture solution without hormone supplements (Gibco, Grand Island,N.Y.). The acetoxymethylester of Fura-2 (Fura-2/AM) and thepentapotassium salt Fura-2 were purchased from Molecular Probes (Eugene,Oreg.). To obtain external calibration standards for the dye, thepentapotassium salt Fura-2 was used at 15 μM in solution with 150 mMKCl, 20 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazine- ethanesulfonicacid (HEPES), and either 5 mM CaCl₂ or 2 mM EGTA. For intracellularcalcium studies NaCl Ringer solution at 25° C. was employed, containingthe following (in millimolar): 150 NaCl, 5 KCl, 5 D glucose, 10 HEPES, 2CaCl₂, 2 MgCl₂, adjusted to pH 7.4. For studies using solutions free ofcalcium, 2 mM EGTA replaced the CaCl₂.

EXAMPLE 1 Cell Culture

Freshly excised human nasal epithelium from normal or cystic fibrosissubjects was grown in primary culture in F-12, hormone-supplementedmedium in accordance with known procedures. See R. Wu et al., Am. Rev.Respir. Dis. 132, 311-320 (1985). For [Ca²⁺ ]_(i) measurements, cultureswere grown on non-fluorescent vitrogen substrates on glass coverslips.Cultures for electrophysiologic studies were grown to confluence onpermeable collagen matrix supports (CMS), allowing addition of agoniststo the basolateral or apical surface of the epithelial sheet.

EXAMPLE 2 Bioelectric Studies

Confluent monolayers of primary human nasal epithelium were mounted inmodified Ussing chambers in accordance with known procedures. See, e.g.,R. Boucher et al., J. Physiol. (Lond.) 405, 77-103 (1988); M. Knowles etal., Science 221, 1067-1070 (1983). The studies described below wereperformed under short circuit current conditions at 37° C., and in somestudies, amiloride was added to the apical bath (10⁻⁴ M) to blocktransepithelial sodium transport. Changes in transepithelial potentialdifference (V_(t)), resistance (R_(t)) and short circuit current(I_(sc)) were measured in response to addition of various nucleotides.Each cultured preparation was exposed to only one concentration of anagonist on either the basolateral or apical surface to avoid thetachyphylaxis observed in preliminary cumulative dose response studies.To construct concentration-effect relationships of responses tonucleotides, it was assumed that the same maximum response to an agonistcould be induced from each tissue culture preparation from the sameindividual.

EXAMPLE 3 Measurements of Intracellular Calcium

Primary human nasal epithelial cells grown to confluence on vitrogencoated coverslips were loaded with a final concentration of 3 μMFura-2/AM at 37° C. for 30 minutes. The cells were then washed in NaClRinger and mounted in a chamber for measurements of fluorescence. Toreduce the rate of leakage of Fura-2 from the cell into theextracellular space and avoid time-dependent compartmentalization of theprobe, all measurements of [Ca²⁺ ]_(i) were performed at 25° C. At thistemperature, no vesicular bright spots indicative ofcompartmentalization of the probe were observed.

Measurements of [Ca²⁺ ]_(i) in single human nasal epithelial cells wereobtained with a modular microspectrofluorimeter (SPEX Industries, Inc.,Edison, N.J.) attached to a Zeiss Axiovert IM 35 microscope. The systemwas equipped with a xenon lamp, beam splitter, two monochromators and arotating chopper mirror that permitted excitation of cell fluorescenceat alternating wavelengths of 340 and 380 nm (emission ≧450 nm). Thefluorescent signal from a single cell was measured with a photometerequipped with a pinhole (spot diameter of 3-5 μm) that excluded signalsfrom adjacent cells.

After agonist was added, the fluorescent signal was quenched by a NaClringer solution containing 1.5×10⁻⁴ M digitonin and 10⁻³ M MnCl₂. Theremaining signal at each excitation wavelength, equivalent to thebackground fluorescence in non loaded cells, was subtracted from datafrom Fura-2/AM loaded cells before the ratio (340 nm/380 nm) was taken.The 340 nm/380 nm ratio was converted to an actual [Ca²⁺ ]_(i)measurement by using the external calibration standards and the formuladerived by G. Grynkiewicz et al., J. Biol. Chem. 260, 3440-3450 (1985),used with dual wavelength measurements: [Ca²⁺ ]_(i) =K [(R_(x)--R_(o))/(R_(s) --R_(x))], with R_(o) and R_(s) representing the ratiosat 0 Ca²⁺ and saturating Ca²⁺, respectively. R_(x) represents theexperimental ratio. K is K_(d) (F_(o) /F_(s)), with K_(d) =1.57×10⁻⁷ Mat 25° C. as the effective dissociation constant for Fura-2, and F_(o)and F_(s) represent the fluorescence intensities at 380 nm with zero andsaturating Ca²⁺, respectively.

EXAMPLE 4 Normal Human Nasal Epithelium--Ion Transport

The effects of extracellular ATP on normal human nasal epithelium wereinvestigated employing bioelectric measurements of ion transportactivity when the nucleotide was applied to either the apical orbasolateral membrane. ATP rapidly stimulated an increase in I_(sc) whenapplied to either surface (data not shown). In general, the change inI_(sc) induced by application of ATP to the apical or the basolateralside returned to baseline or below within 5 minutes after addition ofagonist. Oscillations in I_(sc) following ATP were frequently observed.Apical pretreatment with amiloride (10⁻⁴ M) removes active Na⁺absorption as a component of the I_(sc) so that the residual I_(sc)reflects a Cl⁻ secretory current (R. Boucher et al., J. Clin. Invest.78, 1245-1252 (1986); N. Willumsen et al., Am. J. Physiol. 256,C1033-C1044 (1989)).

A typical Cl⁻ secretory response of human nasal epithelium was foundwhen ATP is applied to amiloride-pretreated tissues (data not shown).Following the initial peak, the ATP induced increase in I_(sc) afterbasolateral addition to amiloride-pretreated tissues returned tobaseline within 5 minutes. In contrast, most tissues treated with ATP onthe apical surface following amiloride pretreatment exhibited prolonged(>10 minutes) increases in I_(sc) above baseline levels.

A concentration-effect relationship was found when ATP was applied tonormal human nasal epithelium under basal conditions (data not shown).Comparisons were made between responses of tissues from different donorsbased on the peak change in I_(sc) following ATP application. The curvedescribes the mean peak change in I_(sc) in response to log increasingconcentrations of ATP applied to the apical or basolateral surface. Theeffectiveness of the nucleotide is approximately equal when applied tothe apical or basolateral surface between 10⁻⁷ and 10⁻⁴ M. A largeincrease in I_(sc) is seen with 10⁻³ M ATP applied to the basolateralmembrane that is not seen with apical application.

Concentration-effect relationships were also found for ATP applied tothe apical or basolateral membrane of amiloride-pretreated tissues (datanot shown). Again, the nucleotide's effect on ion transport was examinedas the mean peak change in I_(sc) after ATP application. The change inion transport induced by ATP in amiloride-pretreated tissues isroutinely smaller than that observed in tissues in the basal state. Thepotency and effectiveness of ATP in amiloride-treated tissues aresimilar whether applied to the apical or basolateral membrane and thelog concentration-effect curves are sigmoidal in character, with EC₅₀values of approximately 1-2×10⁻⁵ M.

The purinergic receptor subtype(s) linked to regulation of ion transportof human nasal epithelium were characterized by obtainingconcentration-effect relationships for a variety of purine andpyrimidine agonists in preparations derived from normal and CF patients.We characterized receptor subtype(s) on the basolateral surface bymeasuring nucleotide effect on basal Na⁺ transport rates. Becausetherapies designed to induce Cl⁻ secretion might best be delivered bythe aerosol route, receptor subtype characterization on the apicalbarrier was performed in the presence of amiloride. The effect ofextracellular nucleotides on ion transport is reported as the percentchange in I_(sc) from control values when applied to the basolateral orapical surface of the culture. Basal pre-agonist currents were similarfor tissues in each concentration group.

Under the culture conditions employed in these studies, the P₁ receptoragonist adenosine, following preincubation of tissues with dipyridamole[10⁻⁶ M] to block adenosine uptake, induced only small and variablechanges in I_(sc) compared to ATP (compare with FIGS. 1 and 2, below).Addition of adenosine to the apical surface of amiloride-pretreatedhuman nasal epithelium at 10⁻⁵ M (n=7) or 10⁻⁴ M (n=8) induced anincrease of 10±7 or 10±6 percent, respectively, whereas addition of thesame doses to the basolateral barrier (n=8, each dose) raised I_(sc) byless than 5 percent. These findings suggest that the activation of P₁receptors contributes little to the measured effects of extracellularlyapplied ATP on ion transport. Therefore, we focused on agonists thatinteract with P₂ receptors in the regulation of ion transport and [Ca²⁺]_(i) mobilization in human nasal epithelium.

FIG. 1 illustrates the concentration-effect relationships of agonistsapplied to the basolateral surface of airway epithelium. In FIG. 1, meanbasal I_(sc) in each set of experiments were (in μA cm⁻²): ATP (◯),69±5, range 55-80, n=5 (n=3-11 at each agonist concentration); UTP (),66±9, range 21-158, n=9 (n=3-23); 2MeSATp (□), 48±6,range 35-65, n=6(n=3-17); ATPγS (), 47±8, range 28-72, n=3 (n=3); ADPβS (Δ), 49±6,range, 36-65, n=3 (n=3); αβMeATP (), 51±12, range 28-66, n=3 (n=3);βγMeATP (), 79±21, range 59-100, n=3 (n=3). Compared to theconcentration-effect curve of ATP, agonists that stimulate P_(2x)receptors (αβMeATP and βγMeATP) induced little change in ion transportrates. The observed rank order of potency for agonists thatsignificantly increased I_(sc) was 2MeSATP>UTP≧ATP>ATPγS>ADP>ADPβS. Atconcentrations of ATP, UTP, ATPγS or ADPβS between 10⁻⁷ and 10⁻⁴ M, therelationship between log agonist concentration and observed responseswas a curve of sigmoidal character. The concentration-effect curve ofthese agonists was biphasic in character when the effect on I_(sc) of10⁻³ M drug was considered.

The concentration-effect relationships for nucleotides added to theapical surface of amiloride-pretreated tissues are shown in FIG. 2. InFIG. 2, mean post amiloride I_(sc) in each set of experiments were (inμA cm⁻²): ATP (◯), 13±1, range 9-16, n=8 (n=3-11 at each agonistconcentration); UTP (), 12±1, range 11-15, n=5 (n=3-17); ATPγS (), 12±1,range 8-15, n=3 (n=3); 2MeSATP (□), 16±2, range 12-21, n=3 (n=3-7);ADPβS (Δ), 15±1, range 13-16, n=3 (n=3); βγMeATP (), 14±0, range 8-19,n=3 (n=3); αβMeATP (), 11±0, range 7-14, n=3 (n=3). Compounds reportedto be effective P_(2x) receptor agonists stimulated little change in iontransport by airway epithelium. Those reported to be effective P_(2y) orUTP sensitive receptor agonists stimulated Cl⁻ secretion with thefollowing rank order of potency: ATP≧UTP>ATPγS>ADP>2MeSATP>ADPβS.

EXAMPLE 5 Normal Human Nasal Epithelium--Intracellular Calcium

Based on studies in other epithelia indicating that regulation of [Ca²⁺]_(i) by purinergic receptors initiates changes in ion transport rates(G. Kimmich and J. Randles, Am. J. Physiol. 243, C116-C123 (1982)), weasked whether ATP regulated [Ca²⁺ ]_(i) in single human nasal epithelialcells using intracellular Ca²⁺ sensitive fluorescent dye. Extracellularapplication of ATP induced an immediate increase in [Ca²⁺ ]_(i) levelsthat decreased over 1 to 2 minutes to a prolonged plateau (data notshown). Exposure to ATP in Ca²⁺ -free medium resulted in an initialsharp increase in [Ca²⁺ ]_(i) which returned to baseline over a twominute period with no plateau phase observed (data not shown). Return ofthe cells to a Ca²⁺ -containing solution resulted in restoration of theplateau phase in the Ca²⁺ response to ATP.

To investigate whether changes in [Ca²⁺ ]_(i) might be related toregulation of ion transport, receptor characterization was performed bymeasuring changes in [Ca²⁺ ]_(i) in response to a number of nucleotidedrugs. Concentration-effect curves for agonists active at P_(2x), P_(2y)subtype or UTP sensitive receptors and other purine or pyrimidinereceptor agonists were generated, measuring mean change in [Ca²⁺ ]_(i)in response to agonist concentration. Data are shown in FIG. 3, whichgives log concentration-effect relationships of purinergic andpyrimidinergic compounds on [Ca²⁺ ]_(i) (mean change in [Ca²⁺ ]_(i) overbasal levels). FIG. 3A shows a comparison of agonists which bind P_(2x),P_(2y) or UTP sensitive receptors, mean basal [Ca²⁺ ]_(i) in each set ofexperiments were (in nM): UTP (◯), 81±8, range 48-113, n=5 (n=3-6);ATPγS (), 116±11, range 85-151, n= 4 (n=3-6); ATP (□), 61±3, range51-70, n=8 (n=3-8 at each agonist concentration); 2MeSATP (), 123±19range 95-180, n=3 (n=3); ADPβS (Δ), 91±17, range 74-107, n=3 (n=3);αβMeATP (), 82±16, range 50-99, n=3 (n=3); βγMeATP (), 68±4, range61-74, n=3 (n=3). FIG. 3B shows a comparison of other purine agonistswith response stimulated by ATP (◯), mean basal [Ca²⁺ ]_(i) in each setof experiments were (nM): GTP (), 117±13, range 92-153, n=3 (n=3);AMPPNP (□), 137±50, range 74-235, n=3 (n=3); ITP (), 103±10, range85-121, n=3 (n=3); ADP (Δ), 94±16, range 75-127, n=3 (n=2-3); AMP (),111±28, range 69-189, n=3 (n=3); ATPαS (), 89±2, range 87-91, n=1 (n=2).FIG. 3C shows a comparison of UTP (◯) with other pyrimidine agonists,mean basal [Ca²⁺ ]_(i) (in nM): 5BrUTP (), 110±13, range 82-152, n=3(n=2-3); UDP (□), 89±2, range 84-92, n=3 (n=3); CTP (Δ), 83±10, range69-102, n=3 (n=3); UMP (), 78±14, range 64-91, n=2 (n=2).

ATP, UTP and ATPγS were the most effective agonists. Classical P_(2x)(αβMeATP and βγMeATP) and P_(2y) (2MeSATP and ADPβS) receptor agonistshad little effect (FIG. 3A) as did other analogs of ATP and ADP (FIG.3B). 5BrUTP was essentially as effective as UTP for stimulation of Ca²⁺mobilization (FIG. 3C).

EXAMPLE 6 Cystic Fibrosis Nasal Epithelium

Availability of Cystic Fibrosis (CF) tissues is limited, and ourinvestigation of effects of nucleotides was restricted to examiningregulation of Cl⁻ secretion rates and [Ca²⁺ ]_(i) levels by ATP and UTP.Only small changes in Cl⁻ secretion (amiloride-resistant I_(sc)) wereobserved in CF tissues following basolateral addition of ATP [14±3maximum mean % change in I_(sc) (n=6)] compared with normal tissues[51±8 maximum mean % change in I_(sc) (n=6)]. Apical administration ofATP following blockade of Na+ absorption with amiloride resulted in twodistinct patterns of response in tissues from CF subjects. Data aregiven in FIGS. 4A, 4B, 4C and 5.

FIGS. 4A, 4B, 4C provides representative bioelectric tracings of effecton I_(sc) of extracellular ATP (10⁻⁴ M) or UTP (10⁻⁴ M) applied to theapical surface of amiloride-pretreated (10⁻⁴ M) CF human nasalepithelium. (A) Cl⁻ secretion in response to ATP (post-amiloride I_(sc)=13 μA.cm⁻²). (B) I_(sc) response to ATP of opposite polarity(post-amiloride I_(sc) =19 μA.cm⁻²) (C) Cl⁻ secretory response to UTP(post-amiloride I_(sc) =5 μA.cm⁻²).

FIG. 5 shows log concentration-effect curves for changes in I_(sc) frombasal levels when ATP or UTP are applied to the apical surface ofamiloride-pretreated (10⁻⁴ M) CF tissues. Mean post amiloride I_(sc) ineach set of experiments were (in μA.cm⁻²): ATP [Grp A (), Grp B (□)],13±1, range 3-31, n=4 (n=4-13 at each agonist concentration); UTP (◯),12±2, range 8-14, n=3 (n=3). s.e. of each data point is ≦13% of thenormalized maximum response.

Most tissues exhibited an increase in I_(sc) over basal levels after ATP(FIG. 4A), suggesting stimulation of Cl⁻ secretion. However,approximately 30% of CF tissues tested responded with a change toopposite polarity of I_(sc) following ATP (FIG. 4B), suggesting asecretion dominated by cations in these preparations (Bean and D. Friel,In: Ion Channels, Vol. 2, 169-203 (T. Narahashi, Ed. 1990)). UTP appliedto the apical surface routinely stimulated increases in Cl⁻ secretion inCF tissues (FIG. 4C). Mean peak change in I_(sc) from post-amiloridebasal levels in response to ATP or UTP application was measured inindividual CF tissues to obtain the concentration-effect relationshipsillustrated in FIG. 5.

Increases in [Ca²⁺ ]_(i) in CF tissues were also observed after additionof ATP or UTP. Data are given in FIG. 6 which shows the logconcentration-effect relationships of the effect of ATP and UTP on [Ca²⁺]_(i) (change from basal levels) in single CF nasal epithelial cells.Mean basal [Ca²⁺ ]_(i) (in nM): ATP (◯), 65±6, range 44-86, n=3 (n=3 ateach agonist concentration); UTP (), 67±3, range 56-79, n=3 (n=3-4).s.e. of each data point is ≦8% of the normalized maximum response. Thesimilar potency and effectiveness of ATP and UTP in these tissuessuggests the presence of a P₂ receptor type sensitive to UTP on CFepithelium.

The foregoing is illustrative of the present invention, and not to beconstrued as limiting thereof. For example, those skilled in the artwill appreciate that minor substitutions can be made to the activecompounds described herein, without departing from the presentinvention. Accordingly, the invention is defined by the followingclaims, with equivalents of the claims included therein.

That which is claimed is:
 1. A method of hydrating mucous secretions inthe lungs of a subject in need of such treatment, comprisingadministering to the lungs of the subject a compound of Formula I below,or a pharmaceutically acceptable salt thereof, in an amount effective tohydrate lung mucous secretions: ##STR2## wherein: X₁, X₂, and X₃ areeach independently selected from the group consisting of OH and SH;R₁ isselected from the group consisting of O, imido, methylene, anddihalomethylene; and R₂ is selected from the group consisting of H andBr.
 2. A method according to claim 1, wherein said compound is deliveredby administering an aerosol suspension of respirable particles comprisedof said compound to the lungs of said subject.
 3. A method according toclaim 2, wherein said particles are selected from the group consistingof solid particles and liquid particles.
 4. A method according to claim2, wherein said aerosol is comprised of particles having a particle sizewithin the range of about 1 to 10 microns.
 5. A method according toclaim 1, wherein said compound is administered in an amount sufficientto achieve concentrations thereof on the airway surfaces of said subjectof from about 10⁻⁷ to about 10⁻³ Moles/liter.
 6. A method according toclaim 1, further comprising concurrently administering amiloride to saidsubject in an amount effective to inhibit the reabsorption of water fromlung mucous secretions.
 7. A method according to claim 1, wherein X₂ andX₃ are OH.
 8. A method according to claim 1, wherein R₁ is oxygen.
 9. Amethod according to claim 1, wherein R₂ is H.
 10. A method according toclaim 1, wherein said compound is selected from the group consisting ofuridine 5'-triphosphate, uridine 5'-O-(3-thiotriphosphate), and thepharmaceutically acceptable salts thereof.
 11. A method of treatingcystic fibrosis in a human subject in need of such treatment, comprisingadministering by inhalation an aerosol suspension of respirableparticles to the respiratory system of said subject, said particlescomprised of a compound of Formula I below, or a pharmaceuticallyacceptable salt thereof: ##STR3## wherein: X₁, X₂, and X₃ are eachindependently selected from the group consisting of OH and SH;R₁ isselected from the group consisting of O, imido, methylene, anddihalomethylene; and R₂ is selected from the group consisting of H andBr; in an amount effective to hydrate retained lung mucous secretions inthe lungs of said subject, whereby the retained mucous secretions aremore easily transported from the lungs via mucociliary action.
 12. Amethod according to claim 11, wherein said particles are selected fromthe group consisting of solid particles and liquid particles.
 13. Amethod according to claim 11, wherein said aerosol is comprised ofparticles having a particle size within the range of about 1 to 10microns.
 14. A method according to claim 11, wherein said compound isadministered in an amount sufficient to achieve concentrations thereofon the airway surfaces of said subject of from about 10⁻⁷ to about 10⁻³Moles/liter.
 15. A method according to claim 11, further comprisingconcurrently administering amiloride to said subject in an amounteffective to inhibit the reabsorption of water from lung mucoussecretions.
 16. A method according to claim 11, wherein X₂ and X₃ areOH.
 17. A method according to claim 11, wherein R₁ is oxygen.
 18. Amethod according to claim 11, wherein R₂ is H.
 19. A method according toclaim 1, wherein said compound is selected from the group consisting ofuridine 5'-triphosphate, uridine 5'-O-(3-thiotriphosphate), and thepharmaceutically acceptable salts thereof.
 20. A pharmaceuticalformulation comprising amiloride in an amount effective to inhibit thereabsorption of water from lung mucus secretions and a compound ofFormula I below, or a pharmaceutically acceptable salt thereof, in anaerosolizable form to be delivered to the lungs of a subject in anamount effective to hydrate lung mucous secretions, in apharmaceutically acceptable carrier: ##STR4## wherein: X₁, X₂, and X₃are each independently selected from the group consisting of OH andSH;R₁ is selected from the group consisting of O, imido, methylene, anddihalomethylene; and R₂ is selected from the group consisting of H andBr.
 21. A pharmaceutical formulation according to claim 20, wherein saidcarrier is selected from the group consisting of solid carriers andliquid carriers.
 22. A pharmaceutical formulation according to claim 20,wherein X₂ and X₃ are OH.
 23. A pharmaceutical formulation according toclaim 20, wherein R₁ is oxygen.
 24. A pharmaceutical formulationaccording to claim 20, wherein R₂ is H.
 25. A pharmaceutical formulationaccording to claim 20, wherein said compound is selected from the groupconsisting of uridine 5'-triphosphate, uridine5'-O-(3-thiotriphosphate), and the pharmaceutically acceptable saltsthereof.
 26. A composition according to claim 20, wherein said compoundof Formula I is in the form of respirable particles ranging in size from1 to 10 microns.